Single-Cell Analysis of Gene Regulatory Networks in the Mammary Glands of P4HA1-knockout mice

Prolyl hydroxylation, catalyzed by collagen prolyl-4 hydroxylase (P4H), is a crucial post-translational modification involved in collagen biosynthesis. P4HA1, an isoform of P4H, plays a prominent role in stabilizing hypoxia-inducible factor-1α (HIF-1α). P4HA1 is frequently upregulated in highly aggressive triple-negative breast cancer and has been implicated in tumor progression, metastasis, and chemoresistance. In this study, we investigated the role of P4HA1 in mouse mammary glands by analyzing gene regulatory networks (GRNs) in basal epithelial cells across two mouse groups: control (5Ht) and P4HA1-knockout (6Ho) mice. Specifically, we employed a single-cell network inference approach, integrating single-cell RNA sequencing with the SCENIC pipeline, and incorporated multiple validation strategies to construct gene regulatory networks (GRNs) specific to basal epithelial cells from each mouse group. Despite the inherent challenges of single-cell data, our approach identified robust and reproducible GRN patterns across both mouse groups. Based on these patterns, we identified subclusters of basal epithelial cells with similar regulatory profiles across the two mouse groups and a unique subcluster in the control mice with a distinct regulatory pattern absent in the P4HA1-deficient 6Ho mice. This unique subcluster exhibited concurrent activation and potential crosstalks between stem cell development and inflammatory response pathways, underscoring the crucial role of P4HA1 in regulating these biological processes linked to cancer progression. We validated these findings through multiple approaches, including experimental validation. Given that the loss of P4HA1 disrupts the interplay between stem cells and inflammation, our results suggest that targeting P4HA1 may offer a promising therapeutic strategy for breast cancer treatment. Author Summary In this study, we aimed to understand how the P4HA1 gene affects the development and functions of mammary gland cells in mice using a single-cell network approach. P4HA1 plays an important role in collagen production, which is essential for maintaining the structure of normal tissues; however, it also helps stabilize the hypoxia-inducible factor-1α (HIF-1α) protein that responds to low oxygen levels, a condition often found in various cancers. We generated and analyzed single-cell RNA sequencing data to understand the regulatory roles of P4HA1 in two groups of mice: one with a single functional allele of P4HA1 and the other with the P4HA1 gene knocked out. We constructed gene regulatory networks for basal epithelial cells in both groups using the SCENIC pipeline. Due to the inherent complexity and noise within single-cell data, we employed multiple validation strategies to ensure that the identified regulatory patterns in the networks across both mouse groups were robust and reproducible. In particular, we found that the P4HA1 gene plays a crucial role in pathways related to stem cell development and inflammation, which are essential for both tissue growth and repair, as well as cancer progression. One unique subcluster of basal epithelial cells found only in control mice with P4HA1 showed simultaneous activation of stem cell and inflammatory processes, suggesting that this gene plays a significant role in regulating the communication between these vital cellular processes. Our work provides new insights into how P4HA1 might serve as a therapeutic target to slow down or prevent the progression of aggressive cancers, such as triple-negative breast cancer, where treatment options are currently limited, leading to a high mortality rate.